Despite improvements in short-term results through the use of new immunosuppressive agents, long-term allograft survival has not improved significantly over the past decade, due predominantly to chronic rejection but also to infection and post-transplant malignancies, all attributable to chronic immunosuppression. Therefore, induction of specific transplantation tolerance, which might eliminate many of these limitations of conventional immunosuppressive therapy, remains a major goal. Our previous studies have demonstrated that the establishment of mixed lymphohematopoietic chimerism provides an effective means for the induction of long-term transplantation tolerance across major histocompatibility barriers in mice. Based on such observations, we successfully developed a non-myeloablative perioperative conditioning regimen that is capable of reliably inducing mixed chimerism and renal allograft tolerance in MHC mismatched cynomolgus monkeys. However, extension of this approach to heart transplantation has proved to be more difficult. In addition to requiring modification of the conditioning regimen to accommodate cadaveric donors, the heart allograft itself has proved to be less capable than the kidney of inducing tolerance. With the mixed chimerism approach, this is likely due to the limited engraftment of donor marrow that has been achieved. Other possibilities might include expression by the heart of novel antigens to which tolerance was not induced or better expression by the kidney of cells essential to tolerance induction (being explored in Project 0013). Therefore, the major goal of this proposal is to modify our current regimen based upon better understanding of the mechanisms of tolerance that is induced by the conditioning protocol in order to design a clinically applicable regimen for heart transplantation. Specifically, we will: 1) evaluate various combinations of costimulatory blockage (with anti-CD154, CTLA4-Ig and anti CD28 monoclonal antibody, 2) clarify the mechanisms of tolerance induced by costimulatory blockade and mixed chimerism, 3) develop modifications of the preparative regimen which will extend its applicability to cadaver-donor recipients as well as recipients already being treated with conventional immunosuppression. The analyses planned should provide valuable information for rationally modifying the conditioning regimen in order to extend this approach to clinical allotransplantation of the heart.